Scientia Horticulturae 123 (2010) 336–342

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Scientia Horticulturae

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Effects of nitrogen deficiency on , status and production in two olive cultivars ‘Meski’ and ‘Koroneiki’

O. Boussadia a,*, K. Steppe b, H. Zgallai d, S. Ben El Hadj c, M. Braham a, R. Lemeur b, M.C. Van Labeke e a Institute of the Olive Tree Station of Sousse, 40 Rue Ibn Khouldoun, 4061 Sousse, Tunisia b Laboratory of Ecology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium c Agronomic National Institute of Tunisia, 43 Avenue Charlnicol, 1082 cite´ El Mahrajen Tunisia d Bayer BioScience N.V. Nazarethsesteenweg 77, B-9800 Astene (Deinze) Belgium e Faculty of Bioscience Engineering, Department of Plant Production, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

ARTICLE INFO ABSTRACT

Article history: The effects of nitrogen deficiency on CO2 assimilation, carbohydrate content and biomass were studied in Received 9 February 2009 two olive (Olea europaea L.) cultivars (‘Meski’ and ‘Koroneiki’). One-year-old were grown in pots Received in revised form 23 September 2009 and subjected to four nitrogen levels for 58 days. Accepted 29 September 2009 Nitrogen-deficient plants had significant lower leaf nitrogen and chlorophyll a contents. They also showed a significant reduction in their photosynthetic capacity. A tolerance difference between cultivars Keywords: was observed: ‘Meski’ proved to be more efficient in maintaining CO2 assimilation rates than ‘Koroneiki’ Carbohydrate under nitrogen deficiency, which was reflected by increased photosynthetic nitrogen use efficiency. Nitrogen deficiency Accumulation of , especially starch, mannitol, sucrose and glucose, was observed in Olive tree Photosynthesis nitrogen-deficient . This indicates that both the high carbohydrate and the low nitrogen content Pigments inhibit photosynthesis in nitrogen-deprived olive plants. Total biomass was strongly reduced (mainly Photosynthetic nitrogen use efficiency caused by a decrease in leaf dry weight) under nitrogen deficiency for both cultivars, but :shoot ratio was hardly affected. Elongation of fine was enhanced in ‘Koroneiki’ under severe nitrogen- deprivation. ß 2009 Elsevier B.V. All rights reserved.

1. Introduction N shortage results in a marked decrease in plant photosynthesis in many crops. This is to be expected, because more than half of the Modern farming requires increasingly fine management of total leaf N is allocated to the photosynthetic apparatus (Makino nitrogen (N) fertilisation due to economical and environmental and Osmond, 1991). Photosynthetic capacity and total amount of constraints. Moreover, excess fertiliser application is expensive leaf N per unit leaf area are usually correlated (Field and Mooney, and leads to N losses by leaching with negative impacts on the 1986; Sage and Pearcy, 1987; Walcroft et al., 1997). environment. Fertilisation must, however, be sufficient to provide There is nowadays clear evidence that N deficiency induces an optimal final yield and the desired product quality. Thus, the sink limitation within the whole plant due to decreased growth required level of accuracy for fertilisation practices is within 10% (Paul and Foyer, 2001). This leads, in turn, to feedback down- (Gastal and Lemaire, 2002). regulation of photosynthesis. N deficiency results in accumula- Olive tree (Olea europaea L.) is one of the major crops in the tion of carbohydrates (sugars and starch) in the leaves, higher Mediterranean basin with a cultivated area of about 8.2 Mha. levels of carbon allocated to the roots and an increase in root-to- Traditionally, fertilisers as well as other crop inputs are applied to shoot biomass ratio (Hirai et al., 2004; Scheible et al., 2004; olive orchards without considering spatial variability in field Remans et al., 2006). N deficiency therefore affects, to various characteristics. Such agricultural management might be inefficient extents, primary photosynthesis, sugar metabolism and/or due to under-application or over-application of field inputs in carbohydrate partitioning between source and sink tissues (Paul specific orchard areas. Under-treated zones will not reach and Driscoll, 1997; de Groot et al., 2003; Scheible et al., 2004). optimum yield levels whereas in over-treated ones there might Although N content in leaves did not correlate with shoot growth, be a higher risk of environmental pollution and reduced cost it was negatively correlated with the proportion of carbon efficiency (Bouma, 1997). allocated to the roots (de Groot et al., 2003; Scheible et al., 2004). Plants indeed constantly sense the changes in their environment and, when mineral elements are scarce, they often allocate a * Corresponding author. Tel.: +216 73236135; fax: +216 73236135. higher proportion of their biomass to the root system (Lawlor E-mail address: [email protected] (O. Boussadia). et al., 2001). This response is a consequence of metabolic changes

0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.09.023 O. Boussadia et al. / Scientia Horticulturae 123 (2010) 336–342 337 in the shoot and an adjustment of carbohydrate transport to the 20N reduced N to 20% N (moderate nitrogen stress with 1 roots. NO3 = 4.79 meq l ) In Tunisia, N deficiency is frequently observed in olive tree 0N reduced N to 0% N (severe nitrogen stress with 1 orchards, and is responsible for considerable losses of productivity NO3 = 0 meq l ) (Braham and Mhiri, 1997). N deficiency is also found to be associated with pistil abortion in olive trees (Morettini, 1950). All other nutrients were provided as specified by the modified Nitrogen excess does not increase yield or vegetative growth Hoagland’s solutions reported in Table 1. (Ferna´ndez-Escobar et al., 2004), but it negatively affects fruit quality or that of derived products, such as olive oil (Ferna´ndez- 2.2. Gas exchange measurements Escobar et al., 2006). However, in current horticultural practice N is often applied in higher amounts than those needed to ensure a Leaf gas exchange measurements were performed weekly on good production (Sa´nchez et al., 1995). the 8th fully expanded leaf counted from the top of the olive tree N fertiliser recommendations for olive trees are traditionally using a LI-6400 portable photosynthesis system (LI-COR Inc., based on soil N status and to a lesser extent on foliar analysis Lincoln, NE, USA), along a period of 58 days (from 24 February till (Boussadia et al., 2006). A rationalization of fertilisation is a 21 April 2008). Measurements took place between 9 and 17 h and prerequisite in the light of the factors up on which productivity were done in 3 replications. The replications were measured, depends, namely photosynthesis, translocation of assimilates, respectively, at 9, 12 and 15 h for each treatment and each growth and production. cultivar in order to cover as such the daily variability. The To gain a better insight in the optimal use of N fertilisers, one photosynthetic active radiation (PAR) was supplied with a 6400- must correctly evaluate the plant’s responses to possible 02B LED light source and photosynthetic light response curves deficiencies. This paper therefore aims at evaluating nutritive N were determined by decreasing light intensity from 2000 to deficiency in two olive tree cultivars ‘Meski’ and ‘Koroneiki’. To our 0 mmol m2 s1 in 9 steps. The temperature inside the leaf knowledge, there is hardly any information available on this cuvette was set to 25 8C, and the CO2 concentration was set to functional approach linking olive tree growth with the N status of 450 mmol mol1. the leaves. To achieve this goal, repeated measurements were Photosynthetic parameters were obtained by fitting the light performed in a greenhouse experiment in order to better under- response curve to the experimental data of each individual plant stand the mechanisms adopted by young olive trees subjected to according to Drake and Read (1981): four levels of N. The effects of N deficiency on chlorophyll concentration, leaf photosynthesis, carbohydrate pools, crop A ðÞI I a A ¼ max c c growth and biomass partitioning were quantified in order to Amax þ ðÞI Ic ac assess the differences in response in both cultivars.

where A and Amax are respectively the net assimilation and the 2 1 2. Materials and methods maximum net assimilation rate (mmol CO2 m s ), ac the 1 quantum efficiency (mmol CO2 (mmol PAR) ), I the light intensity 2 1 2.1. Growth conditions and nitrogen stress treatment and Ic the light compensation point (mmol PAR m s ). 2 1 The dark respiration rate Rd (mmol CO2 m s ) was calculated One-year-old olive trees (Olea L. ‘Meski’ and ‘Koroneiki’) were from the relationship describing the light-limited part of the grown hydroponically in vermiculite (2 l containers) under photosynthesis light response curve, characterized by its linear greenhouse conditions from 22 January till 21 April 2008. Air response to increasing PAR intensities: temperature fluctuated between 20 and 32 8C and the relative humidity of the air ranged between 60 and 70%. Plants were A ¼ acI Rd fertigated with a full-strength modified Hoagland’s solution (EC = 2.5, pH = 6.5) (Table 1) in a hydroponic system (Hartmann For I = Ic, A equals zero and consequently Rd equals acIc from and Brown, 1956). After 34 days of culture, the plants were which ac can be derived. randomly allocated to four groups and each group consisted of 16 After deriving individual plant parameters, mean photosyn- plants (8 plants for each cultivar). Four N levels were provided for thetic parameter values for each N treatment level and each 58 days: cultivar were calculated and used in the analysis.

100N received a full-strength nutrient solution throughout the 2.3. Chlorophyll a concentration 1 experiment (NO3 = 23.96 meq l ) 40N reduced N to 40% N (mild nitrogen stress with Chlorophyll a (Chl a) concentration was measured at weekly 1 intervals according to Moran (1982). Leaf discs (7 mm 19.6 mm/ NO3 = 9.58 meq l ) plant) of randomly chosen fully expanded leaves for each treatment and cultivar were extracted with N,N-dimethylforma- mide (DMF) and absorbance was measured at 664 and 647 nm Table 1 Modified full-strength Hoagland’s nutrient solution (100N) and its adjustments (UV-VIS, Biotek Uvikon XL). Each measurement was done using supplied for the N-deprivation treatments (40N, 20N and 0N). seven plants selected from each treatment and per cultivar.

Macronutrients (g/100l) 100N 40N 20N 0N 2.4. Leaf nitrogen content Ca(NO3)2 109.6 43.84 21.9 0 KNO3 27.4 10.96 5.48 0 Young fully expanded leaves (one leaf per plant) were randomly MgSO 27.4 27.4 27.4 27.4 4 harvested from eight plants in each treatment and combined into a NH4SO4 13.7 5.48 2.74 0

KH2PO4 (MKP) 13.7 13.7 13.7 4.1 composite sample, weighed and analyzed. Leaf tissue nitrogen (N) EDTA Na Fe 4.1 4.1 4.1 4.1 was determined by the Kjeldahl method (Martin-Pre´vel et al., CaCl22H2O 0 48.24 64.34 80.4 1984). Photosynthetic nitrogen use efficiency (PNUE) was calcu- KCl 0 11.91 15.88 19.85 lated as Amax per unit of foliar N content. 338 O. Boussadia et al. / Scientia Horticulturae 123 (2010) 336–342

2.5. Leaf carbohydrate concentration Deprivation to 40N reduced leaf Amax by 16 and 28% for ‘Meski’ and ‘Koroneiki’, respectively, while for 20N leaf Amax was 27 and 49% Sugars were extracted with 80% ethanol at 45 8C, followed by less and for 0N leaf Amax was 34 and 55% less than the control centrifugation at 5000 g for 10 min. Glucose, fructose, mannitol plants. Also Rd was significantly reduced by N deprivation for and sucrose were analyzed using high pH anion-exchange ‘Koroneiki’, while for ‘Meski’ Rd tended not to be affected (Table 2). chromatography with pulsed amperometric detection (Dionex; Finally, ac decreased significantly for both cultivars up to 67 and CarboPac MA1 column with companion guard column; eluent: 31% for ‘Koroneiki’ and ‘Meski’ under 0N, respectively (Table 2).

50 mM NaOH, 22 8C). The remaining ethanol insoluble material A correlation analysis of Amax and N content of the leaves (Fig. 2) was washed twice with ethanol 80% and the residual pellet was showed that for ‘Meski’ Amax was hardly influenced by different treated with HCl 1 M for 2 h at 95 8C for starch hydrolysis. Starch leaf N levels varying between 1.7 and 2.5%. Only for values lower was determined spectrophotometrically at 340 nm by the enzy- than 1.7% Amax slightly decreased. For ‘Koroneiki’, however, a + matic reduction of NADP (UV-VIS, Biotek Uvikon XL). positive correlation between Amax and N content levels up to 2% N was found (R2 = 0.61, p < 0.001) by a non-linear regression. This 2.6. Root, stem and leaf dry weight, and specific leaf weight linear relation is also reflected in the constant PNUE values between the treatments for ‘Koroneiki’ (Fig. 3). For ‘Meski’, At the end of the experiment (after 58 days), three plants were however, no linear relation was found in the data which could randomly harvested per treatment and cultivar. The plants were explain the increase in PNUE compared to control plants (Fig. 3). divided into root, stem and leaf fractions. After measurement of After 58 days of N deprivation, an average significant increase of shoot length, length of the longest root and total leaf area (LI- PNUE of 45% was found in ‘Meski’ for the treatments compared to 3000C portable area meter, Lincoln, NE, USA), the dry weight (DW) 100N. of shoot, leaves and roots was determined after drying at 70 8C for 72 h. Specific leaf weight was calculated as leaf DW per leaf area 3.3. Soluble sugars and starch under nitrogen deficiency according to Syvertsen et al. (1980). For both cultivars N-deficient leaves accumulated significant 2.7. Statistics higher concentrations of starch (Table 3). ‘Koroneiki’ also accumulated higher concentrations of mannitol and glucose under Means and standard deviation were calculated. Analysis of N deprivation. Fructose is hardly present in both cultivars and variance (ANOVA) was performed using SPSS 16.0; when tends to increase under N deprivation for ‘Koroneiki’. Sucrose significant differences occurred, means were separated by the levels in ‘Koroneiki’ showed a moderate though not significant Duncan’s multiple range test at p < 0.05. increase under N deprivation.

3. Results 3.4. Plant growth and dry matter accumulation

3.1. Leaf nitrogen content and chlorophyll a concentration After 58 days of reduced N fertilisation, plant growth was significantly inhibited although cultivar differences were observed Leaf N content averaged 2.0% DW for ‘Koroneiki’ and 1.8% DW (Table 4). for ‘Meski’ under non-limiting N supply (Fig. 1B and E). For ‘Meski’, For ‘Koroneiki’, shoot length did not differ between 100N, 40N reduced N fertilisation was reflected in the leaf N content 35 days and 20N, but plants grown at 0N were 15.6% shorter than control after the start of the treatments resulting in a decrease of plants (p < 0.01). For ‘Meski’, 20N and 0N significantly reduced respectively 51, 58 and 64% for 40N, 20N and 0N in comparison shoot length. The root length of ‘Meski’ was not affected by the with 100N (Fig. 1E) at the end of the experiment. For ‘Koroneiki’, treatments, but for ‘Koroneiki’ root length increased by 52% under leaf N started to decrease 28 days after reducing the N fertilisation. severe N deprivation (0N). Total leaf area as well as number of After 58 days, a decrease of 44, 47 and 55%, respectively, was leaves were reduced under N deprivation for ‘Meski’ compared to observed for 40N, 20N and 0N compared to the 100N treatment control plants. A similar although not significant trend was (Fig. 1B). observed for ‘Koroneiki’ (Table 4). Leaf Chl a concentration averaged 76 mgcm2 for ‘Koroneiki’ Root and stem dry weights were not significantly affected by and 80 mgcm2 for ‘Meski’ for the 100N treatment (Fig. 1A and D). the imposed N deficiency in both cultivars (Table 5). N deficiency After 35 days of withholding N from the nutrient solution for had, however, a pronounced effect on leaf DW of ‘Koroneiki’. The ‘Meski’, the 0N-treated plants started to show significantly lower 40N, 20N and 0N treatments reduced leaf DW respectively by 29, leaf Chl a concentrations compared to the other treatments while it 37 and 28% compared to 100N. The leaf DW of ‘Meski’ also took 49 days for 40N and 20N to decrease significantly from the decreased significantly to 46% for 0N compared to non-limiting N 100N treatment (Fig. 1D). For ‘Koroneiki’, Chl a concentrations fertilisation. started to decrease after 35 days of N deprivation and declined with 33, 32 and 39% respectively for the 40N, 20N and 0N 4. Discussion treatment (Fig. 1A) at the end of experiment. A significant correlation between the decrease of leaf nitrogen In arid regions, drought stress and N deficiency occur mostly content and Chl a concentration was found for the two olive tree simultaneously and the effects on crop production are often 2 2 cultivars (RMeski ¼ 0:65, p = 0.029; RKoroneiki ¼ 0:67, p = 0.025). confounded. In this study, we assessed the effect of leaf N on parameters related to photosynthesis and N supply on growth and 3.2. Leaf photosynthetic response and photosynthetic biomass allocation in 2 olive cultivars grown under non-limiting nitrogen use efficiency water supply. N deficiency affected several components of the carbon The temporal change in photosynthetic capacity under metabolism in the olive trees. The response of leaf photosynthesis decreasing N fertilisation is shown in Fig. 1C and F. After 58 days, is largely dependent on the leaf N content. Low levels of leaf N significant differences between the control and the N-deficient reduced both Chl a concentration and Amax. These results agree plants for leaf Amax were noted in both cultivars (Table 2). with earlier reports in sorghum (Muchow and Sinclair, 1994) and O. Boussadia et al. / Scientia Horticulturae 123 (2010) 336–342 339

Fig. 1. Change in olive tree leaf chlorophyll concentration (Chl a), nitrogen content and net photosynthetic rate (Amax) during the nitrogen (N) deficiency treatment, in ‘Koroneiki’ and ‘Meski’ olive trees. Values are the means along measurement dates and treatments. corn (Wolfe et al., 1988; Settimi and Maranville, 1998; Zhao et al., Sage and Pearcy, 1987; Seemann et al., 1987; Terashima and Evans, 2003). Leaf N content indeed reflects partly the investment in 1988; Sugiharto et al., 1990; Saidana et al., 2009). photosynthetic proteins like Rubisco and the effects of N deficiency In our study N shortage resulted in a marked decrease in plant on Rubisco are often larger than those on Chl a. Effects of N photosynthesis. The relationship between N content and Amax fertilisation on Chl a concentration of plants are also reported showed that the saturation N level for leaf photosynthesis is found elsewhere (Ferrar and Osmond, 1986; Evans and Terashima, 1987; for N content levels above 2% DW and 1.7% DW for ‘Koroneiki’ and

Table 2 2 1 2 1 Effects of nitrogen (N) supply on leaf maximum net assimilation rate (Amax, mmol CO2 m s ), dark respiration (Rd, mmol CO2 m s ) and quantum efficiency (ac, mmol CO2 (mmol PAR)1) of olive leaves measured 58 days after the start of the treatment.

Koroneiki Meski

Amax Rd ac Amax Rd ac

100N 17.7 1.12a 1.17 0.10a 0.065 0.00a 16.20 0.80a 1.12 0.14a 0.040 0.00a 40N 12.8 1.04b 1.02 0.21a 0.029 0.00b 13.60 0.64b 1.43 0.08a 0.030 0.01b 20N 9.1 0.44c 0.53 0.21b 0.025 0.00c 11.75 0.53c 1.38 0.15a 0.030 0.01b 0N 8.0 0.80c 0.36 0.13b 0.020 0.01c 10.60 0.53c 1.56 0.18a 0.030 0.01b

Data are mean SD of three replicates. a, b: significantly different – Duncan (p = 0.05). 340 O. Boussadia et al. / Scientia Horticulturae 123 (2010) 336–342

Fig. 2. Relationship between nitrogen (N) content and leaf maximum net assimilation rate (Amax) under nitrogen deficiency for ‘Koroneiki’ and ‘Meski’ olive trees.

‘Meski’, respectively (Fig. 2). Also, ‘Meski’ maintained better a high leaves in this study may result from the decreased demand for photosynthetic activity under reduced N. carbon in the shoot apical zone due to leaf growth inhibition (leaf Substantial differences between both cultivars were found in area) and leaf initiation, as was the case for ‘Meski’. Because starch is their PNUE. ‘Meski’ showed an increase in PNUE when N leaf the major storage carbohydrate in olive tree leaves (Braham, 1984; content was limited (after 58 days, Fig. 3) while PNUE remained Msallem and Hellali, 1988) and because a corresponding, though not constant for ‘Koroneiki’. The latter indeed showed a linear significant, increase in specific leaf weight in N-deficient leaves was relationship through the origin between leaf Amax and leaf N up observed (Table 5) it might be that starch accumulation contributes to values of 2%, while the following curvilinear relationship to the specific leaf weight. Despite feedback down-regulation of indicated a decrease in PNUE with increasing N availability (Field photosynthesis, the accumulation of excessive amounts of starch, and Mooney, 1986). ‘Meski’, hence, showed an improvement of the however, may disrupt chloroplast structure, leading to lower CO2 N budget in its leaves than ‘Koroneiki’ and might therefore be assimilation and Chl concentration (Cave et al., 1981). This might better adapted to N shortages. also be the case in this study as indicated by the decreased Chl a

Rd provides energy for growth and maintenance of the existing concentration at the end of the experiment (Fig. 1). cell structures (Wilson, 1982). The decrease in Rd of ‘Koroneiki’ N- There often exists a more pronounced down-regulation of deficient plants is more important than the Amax reduction photosynthesis in starch-accumulating species when sink capacity (Table 2). Decreased Rd under N stress was also observed for is limiting (Goldschmidt and Huber, 1992) which is indicated in spinach leaves showing N deficiency symptoms (Bottrill et al., this experiment by reduced shoot growth and leaf area (Table 4). 1970). This might explain that no energy remains for new growth, Previous work on citrus has shown that girdling and defruiting but is directed to maintain reduced leaf biomass for 40N, 20N and may increase carbohydrates, particularly starch, and decrease

0N plants. Rd for ‘Meski’, however, tended to increase (although not photosynthesis (Iglesias et al., 2002). Goldschmidt and Huber significantly) which might explain the higher ability of ‘Meski’ to (1992) investigated the relationship between feedback inhibition maintain the leaf biomass of 40N and 20N plants to the same level of photosynthesis caused by girdling and the type of carbohydrates as 100N. accumulated in the leaves of a wide range of vegetable crops. They The starch content was higher in N-deficient than in control observed that high-invertase-type species next to starch-accumu- leaves for both olive cultivars. This is also observed in other species lating species showed a marked decrease in maximum photo- (Paul and Foyer, 2001). The elevated starch level in N-deficient synthetic capacity. In our research not only starch but also glucose, mannitol and sucrose contents were higher in N-deficient than in control leaves for ‘Koroneiki’. So, the reduction of photosynthesis in N-deficient olive plants is also a direct consequence of sugar next to starch accumulation, because they both exert metabolite feedback regulation (Koch, 2004; Bla¨sing et al., 2005). There is also clear evidence that N deficiency induces sink limitation within the whole plant due to decreased growth (Paul and Driscoll, 1997; Logan et al., 1999; Paul and Foyer, 2001). Decreased plant biomass production due to N shortage was associated with reductions in both leaf area (Saidana et al., 2009) and leaf photosynthetic capacity (Sinclair, 1990). N deficiency suppressed olive shoot growth and dry matter accumulation, especially leaf DW, in both cultivars (Table 5) and this latter was mainly attributed to a reduced total leaf area (Tables 4 and 5). Reduced leaf area was caused by a delay in leaf initiation, but also by leaf abscission visually observed under N deprivation. Olive trees deficient in N improve their ability to acquire this nutrient by altering their carbon partitioning to favour root elongation as was observed for ‘Koroneiki’ under severe N deprivation (Table 4). These young roots were very fine and did Fig. 3. Effect of nitrogen (N) deficiency on photosynthetic nitrogen use efficiency not influence the total root DW. Therefore no effect on root/shoot (PNUE) in ‘Koroneiki’ and ‘Meski’ olive trees calculated 58 days after the start of the N supply treatments. Data are mean values standard deviations of three replicates. ratio was found after 58 day of N-deficient treatment for both a, b: significantly different – Duncan (p = 0.05). cultivars (Table 5). O. Boussadia et al. / Scientia Horticulturae 123 (2010) 336–342 341

Table 3 Effects of nitrogen (N) supply on starch, mannitol, glucose, fructose and sucrose (mg (g DW)1) of olive leaves measured 58 days after the start of the treatment.

Koroneiki Meski

100N 40N 20N 0N 100N 40N 20N 0N

Starch 0.38 0.08d 0.54 0.15c 0.41 0.13b 0.72 0.01a 0.35 0.02c 0.50 0.04b 0.68 0.07a 0.53 0.14ab Mannitol 1.25 0.10c 1.63 0.07a 1.36 0.02c 1.48 0.03b 1.40 0.18a 1.46 0.10a 1.57 0.14a 1.42 0.06a Glucose 1.57 0.25c 1.72 0.37bc 2.25 0.30ab 2.42 0.32a 1.42 0.47a 1.32 0.68a 1.47 0.25a 1.36 0.24a Fructose 0.01 0.01b 0.01 0.01b 0.04 0.01a 0.03 0.01ab 0.04 0.00a 0.03 0.01ab 0.04 0.03b 0.03 0.01ab Sucrose 0.51 0.12a 0.60 0.04a 0.63 0.04a 0.68 0.10a 0.38 0.03a 0.37 0.02a 0.41 0.05a 0.41 0.04a

Data are mean SD of three replicates. a, b: significantly different – Duncan (p = 0.05).

Table 4 Effects of nitrogen (N) deficiency on shoot and root length, leaf area and number of leaves olive plants 58 days after the start of the treatment.

Shoot length (cm) Leaf area (cm2) Number of leaves Root length (cm)

Koroneiki 100N 92 4.0b 1037 396.9a 333 147.8a 32 2.0b 40N 99 9.0b 740 179.7a 299 72.0a 25 4.0c 20N 95 2.2b 587 162.3a 259 94.8a 22 4.0c 0N 78 2.0a 660 37.5a 274 15.6a 67 3.0a

Meski 100N 85 1.0a 388 45.5a 113 13.2a 23 0.5a 40N 82 1.1ab 342 71.1ab 96 1.4ab 22 1.0a 20N 78 5.0b 267 37.5b 91 1.4b 23 0.5a 0N 60 2.0c 168 44.2c 65 17.1c 24 0.3a

Data are mean SD of three replicates. a, b: significantly different – Duncan (p = 0.05).

Table 5 Effects of nitrogen (N) supply on leaf, stem and root dry weight (DW), root/shoot ratio and specific leaf weight of olive plants 58 days after the start of the treatment.

Root DW (g plant1) Stem DW (g plant1) Leaf DW (g plant1) Root/shoot ratio Specific leaf weight (g DW m2)

Koroneiki 100N 13.00 1.41a 12.75 1.06a 16.00 2.83a 0.45 0.01a 143.60 23.12a 40N 12.50 2.60a 13.66 4.37a 11.33 2.57b 0.54 0.27a 153.47 5.59a 20N 11.50 2.12a 14.25 1.06a 10.00 2.12b 0.48 0.15a 165.27 16.50a 0N 10.00 0.71a 12.00 0.71a 11.50 2.12b 0.42 0.00a 171.19 18.12a

Meski 100N 6.16 0.29a 7.16 0.58a 6.83 0.58a 0.44 0.03a 176.72 9.14a 40N 5.25 2.12a 7.25 0.35a 6.00 0.02a 0.40 0.20a 156.67 1.23a 20N 5.25 0.35aa 6.50 0.71a 5.50 0.71a 0.44 0.03a 223.02 17.89a 0N 4.83 1.04a 6.83 0.29a 3.66 1.44b 0.47 0.12a 212.03 30.60a

Data are mean SD of three replicates. a, b: significantly different – Duncan (p = 0.05).

5. Conclusions Department of Plant Production and Mariane Bruggeman of the Research Centre for Ornamental Plants (PCS) for all support and N deficiency caused a decrease in leaf N content, Chl a and technical assistance. The Institution de Recherche et d’Enseigne- carbon assimilation of olive plants, resulting in a lower dry matter ment Superieur Agricole and the Institut de l’Olivier de Sousse for accumulation. Decreased photosynthetic capacity is not only providing the olive trees and technical assistance. The authors also associated with direct effects of N deficiency but also with a wish to thank the Vlaamse Interuniverversitaire Raad (VLIR) for negative feedback mechanism from the leaf carbohydrate pool. doctoral research of the first author and the Research Foundation – Indeed both cultivars showed an important increase in starch and Flanders (FWO-Vlaanderen) for the Postdoctoral Fellow funding for ‘Koroneiki’ also some sugars (glucose and mannitol) accumu- granted to the second author. lated. Total dry matter was reduced mainly caused by a decrease in leaf DW. The shoot:root ratio was hardly affected although root References elongation in N deprived ‘Koroneiki’ was observed. The ecophy- siological and biochemical parameters used in this experiment Bla¨sing, O.E., Gibon, Y., Gu¨ nther, M., Ho¨hne, M., Morcuende, R., Osuna, D., Thimm, O., (A , PNUE, Chl a and carbohydrate accumulation in leaves) Usadel, B., Scheible, W., Stitt, M., 2005. Sugars and circadian regulation make max major contributions to the global regulation of diurnal gene expression in suggest that ‘Meski’ is more efficient than ‘Koroneiki’ under N Arabidopsis. Plant Cell 17, 3257–3281. deficiency. This approach could be further exploited for cultivar Bottrill, D.E., Possingham, J.V., Kriedemann, P.E., 1970. The effect of nutrient selection. deficiencies on photosynthesis and respiration in spinach. Plant Soil 32, 424–438. Bouma, J., 1997. Precision agriculture: introduction to the spatial and temporal Acknowledgements variability of environmental quality. In: Lake, J.V., Bock, G.R., Goode, J.A. (Eds.), Precision Agriculture: Spatial and Temporal Variability of Environ- mental Quality. Wiley, Wageningen, The Netherlands, pp. 5–17. The authors would like to thank Philip Deman and Margot Boussadia, O., Braham, M., Ben Elhadj, S., 2006. Du statut mine´ral de l’olivier dans le Vanneste of the Laboratory of Pant Ecology, Thea Versluys of the semi-aride tunisien. INRAT no 75. 342 O. Boussadia et al. / Scientia Horticulturae 123 (2010) 336–342

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